Specificity grafting of a murine antibody onto a human framework

ABSTRACT

The current invention provides new human variable chain framework regions and humanized antibodies comprising the framework regions. The invention also provides a new method of identifying framework acceptor regions in framework sequences for backmutation to graft a donor sequence to the human framework.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional application No.60/390,033, filed Jun. 17, 2002, which is herein incorporated byreference.

BACKGROUND OF THE INVENTION

Monoclonal antibodies provide powerful diagnostic and therapeutic toolsfor treatment of a variety of disease, e.g., cancer. However, use ofmonoclonal antibodies is limited due to immunogenicity. In order toameliorate these effects, humanization strategies have been devised thatreplace portion of the monoclonal antibodies with human counterparts.

Currant techniques for humanizing antibodies involve selecting thecomplementarity determining regions (CDRs), i.e., the antigen bindingloops, from a donor monoclonal antibody, and grafting them onto a humanantibody framework of known three dimensional structure (see, e.g.,WO98/45322; WO 87/02671; U.S. Pat. No 5,859,205; U.S. Pat. No.5,585,089; U.S. Pat. No. 4,816,567; EP Patent Application 0173494;Jones, et al. (1986) Nature 321:522; Verhoeyen, et al., (1988) Science239:1534 Riechmann, et al. (1988) Nature 332:323; and Winter & Milstein,(1991) Nature 349:293) or performing database searches to identifypotential candidates. In a typical method aided by computer modeling andcomparison to human germline sequences, the antigen binding loops of themonoclonal antibody to be humanized are superimposed onto the bestfitting frameworks. This allows the identification of framework residuesthat are potentially important for the affinity of the antibody.

Humanization efforts are limited, however, by the number of humanframeworks available. This invention meets the need for additional humanframework sequences and provides methods of providing stable humanframework sequences for specificity graftings, and in some embodiments,methods of modifying the sequences to generate stable humanizedantibodies.

BRIEF SUMMARY OF THE INVENTION

This invention relates to humanization of antibodies, specificallyproviding novel biophysically stable human framework sequences that canbe used to humanize antibodies, e.g., single chain Fv (scFv) fragments.Exemplary RFB4 humanized scFv antibodies were constructed using the newhuman framework sequences. Thus, the current invention provides novelhuman framework protein and nucleic acid sequences and humanizedantibodies, e.g., RFB4, that have been generated using the sequences. Inaddition, the invention provides a method of humanizing a donor antibodybased on selecting stable framework sequences by panning a displaylibrary. In some embodiments, the method further comprises steps ofselecting particular residues of the human frameworks for backmutationto donor antibody residues.

In particular embodiments, the invention provides a humanized antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the CDRs of the heavy chain and light chain variableregions are from a donor antibody, and wherein the heavy chain variableregion framework has at least 80%, often 85%, 90%, or 95% identity to aframework comprised by an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. In some embodiments,the framework is comprised by an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

The invention also provides a humanized antibody comprising a lightchain variable region framework that has at least 80%, often 85%, 90%,or 95% identity to a framework comprised by an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ IDNO:15. In some embodiments, the framework is comprised by an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 andSEQ ID NO:15.

In one embodiment, the heavy chain variable region framework has atleast 80% identity, typically at least 90% or 95% identity, to aframework of SEQ ID NO: 1, the light chain variable region has at least80% identity, typically at least 90% or 95% identity, to a framework ofSEQ ID NO:2, the donor CDR sequences are from RFB4, and the antibodyspecifically binds to CD22. This humanized antibody can also comprisesdonor antibody amino acid residues at particular positions, e.g.,V_(H)6, V_(L)3, V_(L)40, V_(L)49, or V_(L)46. In other embodiments, thehumanized antibody can additionally comprise donor antibody amino acidresidues at positions V_(L)36, V_(L)71, V_(H)79, V_(H)40 or V_(H)84. Insome embodiments, the humanized antibody comprises a V_(H) and V_(L) asset forth in SEQ ID NO:21 or SEQ ID NO:22. In other embodiments, thehumanized antibody comprises an amino acid sequence of SEQ ID NO:21 orSEQ ID NO:22.

In another embodiment, the humanized antibody may further comprises anFc region or it may be an scFv.

In another aspect, the invention provides an isolated nucleic acidencoding any of the humanized antibodies set forth above.

In another aspect, the invention provides an immunoconjugate comprisingany of the humanized antibodies set forth above, linked to a detectableor therapeutic moiety. The therapeutic moiety may be, for example, acytotoxic moiety, an enzyme, a cytokine, or a small molecule. Inparticular embodiments, the enzyme is an RNase A family member, such asrapLR1.

The moiety may also be a detectable moiety, such as a fluorescent label,a radioactive, tag, or an enzymatic label that provides a detectablephenotype, e.g., color, in the presence of a substrate.

In another aspect, the invention provides an isolated nucleic acidencoding an immunoconjugate comprising an antibody as set forth aboveand a therapeutic or detectable moiety, wherein the therapeutic ordetectable moiety is a polypeptide.

In another aspect, the invention provides a heavy chain variable (V_(H))chain having at least 80% identity, typically 90%, 95%, or greateridentity, to the framework amino acids residues comprised by an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,and SEQ ID NO:11. In one embodiment, the V_(H) chain has at least 80%identity, typically 90%, 95%, or greater identity, to the frameworkamino acid residues of the V_(H) amino acid sequence set forth in SEQ IDNO: 1 and further, may comprise RFB4 CDRs.

In another aspect, the invention provides a light chain variable (V_(L))chain having at least 80% identity, typically 90%, 95%, or greateridentity, to the framework amino acid residues of a framework comprisedby an amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:14 and SEQ ID NO:15. In one embodiment, the light chain has atleast 80% identity, typically 90%, 95%, or greater identity, to theframework amino acid residues of the V_(L) amino acid sequence set forthin SEQ ID NO:2 and further, may comprise RFB4 CDRs.

In other aspects, the invention provides an isolated nucleic acidencoding a V_(H) chain or V_(L) chain comprising a human framework aminoacid sequence as set forth above.

In another aspect, the invention provides a method of making a humanizedantibody, the method comprising: screening a display library understringent conditions with a screening antigen, thereby generating a poolof antibodies pre-selected for stability and solubility; selecting ahuman V_(H) amino acid sequence or human V_(L) amino acid sequence thathas at least 70% identity to a donor amino acid V_(H) amino acidsequence; and grafting the CDRs from the donor to the selected V_(H) andV_(L) framework sequences.

In some embodiments, the method further comprises aligning the selectedsequences and the donor V_(H) and V_(L) amino acid sequences with knownmouse and human V_(H) and V_(L) sequences; idenitfying candidateresidues in the framework regions of the selected V_(H) and V_(L)sequences that can be backmutated to the donor residues; wherein thecandidate residues selected for backmutation are those residues thatoccur in less than 5% of the sequences analyzed; and backmutating thosecandidate residues that are at sites of the framework regions that areimportant for the binding properties of the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary framework regions of novel human human variableheavy chain and variable light chain regions.

FIG. 2 shows an exemplary sequence alignment of the V_(H) and V_(L)domains of RFB4 and the novel human domains V_(H#)8 and V_(L#)19. Theantigen binding site is defined according to Chothia (Chothia and Lesk,J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989(dotted line)); and Kabat (Kabat and Wu, J Immunol 147:1709-1719,1991(dashed line)); germ, human germline sequences; #8/#19, humanV_(H)/V_(L) acceptor sequence; RFB4, murine V_(H)/V_(L) donor sequence;J gene segments are underlined. (A) “invariant residues” (Kabat and Wu,1991); (B) “key residues” (Chothia et al., 1989) and (C) residues at theV_(H)/V_(L) interface (Chothia et al., 1985) are marked with (+) formatching or (−) for non-matching residues between murine and humansequence, respectively. (D) Residues at core sites as defined by Chothia(Chothia et al., 1998) as invariant (i) residue sites; similar (r)residue sites; surface (s) residues R,K,E,D,Q,N; neutral (n) residuesP,H,Y,G,A,S,T; and buried (b) residues C,V,L,I,M,F,W respectively; b/n,x; s/n, y; non-matching residue sites between murine and human sequenceare marked in bold letters; hum, specificity grafted sequences withmurine back-mutated framework and CDR residues shown in bold letters.All residues are shown in the single letter code and numbered accordingto Kabat (Kabat et al., 1991).

FIG. 3 shows an exemplary equilibrium-binding curves of specificitygrafted scFv monomers. Raji cells were incubated with variousconcentrations of, SGII (closed diamonds), SGIII (closed squares), SGIV(closed triangles), SGV (closed circles), or mAb RFB4 (open circles).Specific binding of antibodies was determined by flow cytometry. Bindingactivity at indicated concentrations is given in percent of the maximalmedian flourescence intensity (MFI). Measurements were performed intriplicate; standard deviations are shown as bars. Binding affinityconstants (K_(d)) were determined by fitting the cell binding data tothe nonlinear regression model according to the Levenberg-Marquardmethod.

FIG. 4 provides exemplary data showing the epitope specificity ofvariants SGIII, SGIV and SGV. Competition of scFv variants with mAb RFB4for binding to CD22⁺ Raji cells was determined by flow cytometry.Results are shown as percent binding inhibition of the mAb (5 nM) whenincubating tumor cells with 200-fold molar excess of scFv variants.

FIG. 5 provides exemplary data showing the serum stability of humanizedscFv's. Constructs SGII (closed diamons), SGIII (closed squares), SGIV(closed triangles), and SGV (closed circles) were incubated at 37° C.for various time points, followed by determination of immunoreactivitywith CD22⁺ Raji cells by flow cytometry.

FIG. 6 provides exemplary data showing the biophysical stability ofspecificity grafted scFv monomers. Analytical size-exclusion FPLC on acalibrated Superdex 75 column was performed before (dashed line) andafter (solid line) incubation of 12 μg/ml scFv in PBS at 37° C. for fivedays.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The current invention provides novel human V_(H) and V_(L) frameworksequences and nucleic acid sequences that encode them. Such sequencesare used, for example, to provide frameworks for grafting CDRs from adonor antibody, e.g., a murine antibody. Thus, an antibody comprising aV_(H) and/or V_(L) framework of the invention with the bindingspecificity of a donor antibody can be created.

Further, the invention provides methods of humanizing an antibody usingstable framework sequences that have not been defined in terms of theirthree dimensional structure, such as the novel human V_(H) and V_(L)sequences provided herein. This method allows the practitioner to selectresidues to back mutate in order to maintain antigen binding propertiesof the donor antibody.

The invention also provides humanized antibodies comprising an RFB4specificity grafted onto the V_(H) and V_(L) framework regions set forthin SEQ ID NOs: 1 and 2.

The humanized antibodies can be used for a variety of therapeutic anddiagnostic purposes described herein.

Definitions

The term “CD22” includes reference to a CD22 antigen present on thesurface of B-cells of a mammal such as rats, mice, and primates,particularly humans. See, e.g. Wilson et al., J. Exp. Med.173(l):137-146 (I 991); Wilson et al., J. Immunol, 150(11):5013-5024(1993). The term “CD22 protein” includes reference to both CD22 andimmunoreactive fragments of CD22. Such CD22 immunoreactive fragmentshave an affinity for a CD22 antigen that is at least 5-fold greater thana non-CD22 control protein.

As used herein, the term “anti-CD22” in reference to an antibody, refersto an antibody that specifically binds CD22.

The term “antibody” refers to a polypeptide encoded by an immunoglobulingene or functional fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Examples of antibody functional fragments include, but are not limitedto, complete antibody molecules, antibody fragments, such as Fv, singlechain Fv (scFv), complementarity determining regions (CDRs), V_(L)(light chain variable region), V_(H) (heavy chain variable region), Fab,F(ab)2′ and any combination of those or any other functional portion ofan immunoglobulin peptide capable of binding to target antigen (see,e.g., Fundamental Immunology (Paul ed., 3d ed. 1993). As appreciated byone of skill in the art, various antibody fragments can be obtained by avariety of methods, for example, digestion of an intact antibody with anenzyme, such as pepsin; or de novo synthesis. Antibody fragments areoften synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, includes antibodyfragments either produced by the modification of whole antibodies, orthose synthesized de novo using recombinant DNA methodologies (e.g.,single chain Fv) or those identified using phage display libraries (see,e.g., McCafferty et al., Nature 348:552-554 (1990)). The term antibodyalso includes bivalent or bispecific molecules, diabodies, triabodies,and tetrabodies. Bivalent and bispecific molecules are described in,e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun(1992) Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber etal. (1994) J Immunol :5368, Zhu et al. (1997) Protein Sci 6:781, Hu etal. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026,and McCartney, et al. (1995) Protein Eng. 8:301.

References to “V_(H)” or a “VH” refer to the variable region of animmunoglobulin heavy chain, including an Fv, scFv , adisulfilde-stabilized Fv (dsFv) or Fab. References to “V_(L)” or a “VL”refer to the variable region of an immunoglobulin light chain, includingof an Fv, scFv, dsFv or Fab.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. The numbering of the light and heavy chainvariable regions described herein is in accordance with Kabat (see,e.g., Johnson et al., (2001) “Kabat Database and its applications:future directions” Nucleic Acids Research, 29: 205-206; and the KabatDatabase of Sequences of Proteins of Immunological Interest, Feb. 22,2002 Dataset) unless otherwise indicated.

The positions of the CDRs and framework regions can be determined usingvarious well known definitions in the art, e.g., Kabat, Chothia,international ImMunoGeneTics database (IMGT), and AbM (see, e.g.,Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures forthe hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human V_(H) segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc,M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(l):207-9 (2001);MacCallum et al, Antibody-antigen interactions: Contact analysis andbinding site topography, J Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

Exemplary framework and CDR sequences for novel human V_(H) and V_(L)regions disclosed herein are shown in FIG. 1. With the exception ofCDR-H1, the antigen binding loops to be grafted onto the human frameworkregions were defined according to Kabat et al. (1991) Sequences ofProteins of Immunological Interest. (NIH Publication No. 91-3242,Bethesda). As residues H26-H32 comprise the structural loop of CDR-H1(Chothia et al, Nature 342:877-883, 1989), residues H26-H35 were appliedas CDR-H1 according to the combined Kabat/Chothia definition of CDR-H1.

“RFB4” refers to a mouse IgGI monoclonal antibody that specificallybinds to human CD22. RFB4 is commercially available under the name RFB4from several sources, such as Southern Biotechnology Associates, Inc.(Birmingham Ala.; Cat. No. 9360-01) and Autogen Bioclear UK Ltd. (Calne,Wilts, UK; Cat. No. AB147). RFB4 is highly specific for cells of the Blineage and has no detectable cross-reactivity with other normal celltypes. Li et al., Cell. Immunol. 118:85-99 (1989). The heavy and lightchains of RFB4 have been cloned. See, Mansfield et al., Blood90:2020-2026 (1997). The amino acid and nucleotide sequences of the RFB4heavy chain are set forth in SEQ ID NO:16 and SEQ ID NO:27,respectively. The amino acid and nucleotide sequences of the RFB4 lightchain are set forth in SEQ ID NO: 17 and SEQ ID NO:28, respectively. TheRFB4 CDRs as designated for the exemplary humanized RFB4 antibodiesdescribed herein are underlined in SEQ ID NOs:16 and 17 and shown inFIG. 2. The CDRs were defined according to Kabat, except for CDR-H1, forwhich the combined Chothia/Kabat definition was applied (comprisingresidues H26-H35.)

A “humanized antibody” refers to a an antibody that comprises a donorantibody binding specificity, i.e., the CDR regions of a donor antibody,typically a mouse monoclonal antibody, grafted onto human frameworksequences. A “humanized antibody” as used herein binds to the sameepitope as the donor antibody and typically has at least 25% of thebinding affinity. An exemplary assay for binding affinity is describedin Example 5. Methods to determine whether the antibody binds to thesame epitope are well known in the art, see, e.g., Harlow & Lane, UsingAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press,1999, which discloses techniques to epitope mapping or alternatively,competition experiments, to determine whether an antibody binds to thesame epitope as the donor antibody. A humanized antibody that comprisesa novel framework region provided in the invention.

A “novel human framework” of the invention refers to the framework of ahuman V_(H) or V_(L) amino acid sequence that has at least 80% identity,often, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, to aframework set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, orSEQ ID NO:15. A “framework” of a V_(H) or V_(L) chain refers to theframework regions of the chain. The term as applied to each chainencompasses all of the framework regions.

A “humanized RFB4” refers to a humanized antibody comprising a humanframework sequence that has the binding specificity of the mouse RFB4grafted to that framework. A CDR of a humanized RFB4 antibody of theinvention has at least 85%, more typically at least 90%, 95%, 96%, 97%,98%, or 99% identity to a CDR of the RFB4 heavy and light chainsequences set forth in SEQ ID NO:16 and SEQ ID NO:17, respectively.Examples of RFB4 CDR variants that retain RFB4 binding specificity areset forth, e.g., in

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor the stabilization of the variable domains without interfering withthe proper folding and creation of an active binding site. A singlechain humanized antibody of the invention, e.g., humanized RFB4, maybind as a monomer. Other exemplary single chain antibodies may formdiabodies, triabodies, and tetrabodies. (See, e.g., Hollinger et al.,1993, supra). Further the humanized antibodies of the invention, e.g.,humanized RFB4 may also form one component of a “reconstituted” antibodyor antibody fragment, e.g., a Fab, a Fab′ monomer, a F(ab)′2 dimer, oran whole immunoglobulin molecule. Thus, a humanized antibody of thepresent invention may further comprise a human Fc region.

“Join” or “link” or “conjugate” refers to any method known in the artfor functionally connecting protein domains, including withoutlimitation recombinant fusion with or without intervening domains,intein-mediated fusion, non-covalent association, and covalent bonding,e.g., disulfide bonding, peptide bonding; hydrogen bonding;electrostatic bonding; and conformational bonding, e.g.,antibody-antigen, and biotin-avidin associations. In the context of thepresent invention, the terms include reference to joining an antibodymoiety to an effector molecule (EM). The linkage can be either bychemical or recombinant means. Chemical means refers to a reactionbetween the antibody moiety and the effector molecule such that there isa covalent bond formed between the two molecules to form one molecule.

The term “effector moiety” means the portion of an immunoconjugateintended to have an effect on a cell targeted by the targeting moiety orto identify the presence of the immunoconjugate. Thus, the effectormoiety can be, for example, a therapeutic moiety, such as a cytotoxicagent or drug, or a detectable moiety, such as a fluorescent label.

A “therapeutic moiety” is the portion of an immunoconjugate intended toact as a therapeutic agent.

The term “therapeutic agent” includes any number of compounds currentlyknown or later developed to act as anti-neoplastic compounds,anti-inflammatory compounds, anti-infective compounds, enzyme activatorsor inhibitors, allosteric modifiers, antibiotics orother agentsadministered to induce a desired therapeutic effect in a patient. Thetherapeutic agent may also be a toxin or a radioisotope, where thetherapeutic effect intended is, for example, the killing of a cancercell.

The terms “effective amount” or “amount effective to” or“therapeutically effective amount” refers to an amount sufficient toinduce a detectable therapeutic response in the subject. Preferably, thetherapeutic response is effective in reducing the proliferation ofcancer cells or in inhibiting the growth of cancer cells present in asubject. Assays for determining therapeutic responses are well known inthe art.

The term “immunoconjugate” refers to a composition comprising anantibody linked to a second molecule such as a detectable label oreffector molecule. Often, the antibody is linked to the second moleculeby covalent linkage.

In the context of an immunoconjugate, a “detectable label” or“detectable moiety” refers to, a portion of the immunoconjugate whichhas a property rendering its presence detectable. For example, theimmunoconjugate may be labeled with a radioactive isotope which permitscells in which the immunoconjugate is present to be detected inimmunohistochemical assays. A “detectable label” or a “detectablemoiety” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical means. Forexample, useful labels include radioisotopes (e.g., ³H, ³⁵S, ³²P, ⁵¹Cr,or ¹²⁵I), fluorescent dyes, electron-dense reagents, enzymes (e.g.,alkaline phosphatase, horseradish peroxidase, or others commonly used inan ELISA), biotin, digoxigenin, or haptens and proteins which can bemade detectable, e.g., by incorporating a radiolabel into the peptide orused to detect antibodies specifically reactive with the peptide.

The term “immunologically reactive conditions” includes reference toconditions which allow an antibody generated to a particular epitope tobind to that epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. Preferably, the immunologically reactive conditions employedin the methods of the present invention are “physiological conditions”which include reference to conditions (e.g., temperature, osmolarity,pH) that are typical inside a living mammal or a mammalian cell. Whileit is recognized that some organs are subject to extreme conditions, theintra-organismal and intracellular environment normally lies around pH 7(i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), containswater as the predominant solvent, and exists at a temperature above 0°C. and below 50° C. Osmolarity is within the range that is supportive ofcell viability and proliferation.

The term “binding specificity,” “specifically binds to an antibody” or“specifically immunoreactive with,” when referring to an epitope, refersto a binding reaction which is determinative of the presence of theepitope in a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular epitope at least two times the background and moretypically more than 10 to 100 times background. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein or carbohydrate. For example, solid-phaseELISA immunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein or carbohydrate. See, Harlow & Lane,ANTI130DIES, A LABORATORY MANUAL, Cold Spring Harbor Press, New York(1988) and Harlow & Lane, USING ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Press, New York (1999), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

“Nucleic acid” and “polynucleotide” are used interchangeably herein torefer to deoxyribonucleotides or ribonucleotides and polymers thereof ineither single- or double-stranded form. The term encompasses nucleicacids containing known nucleotide analogs or modified backbone residuesor linkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs). As appreciate by one of skill in the art, the complementof a nucleic acid sequence can readily be determined from the sequenceof the other strand. Thus, any particular nucleic acid sequence setforth herein also discloses the complementary strand.

“Polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply tonaturally occurring amino acid polymers, as well as, amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refers tocompounds that have the same fundamental chemical structure as anaturally occurring amino acid, i.e., an alpha carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission.

“Conservatively modified variants” applies to both nucleic acid andamino acid sequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

With respect to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologues, and alleles of the invention.

For example, substitutions may be made wherein an aliphatic amino acid(G, A, I, L, or V) is substituted with another member of the group, orsubstitution such as the substitution of one polar residue for another,such as arginine for lysine, glutamic for aspartic acid, or glutaminefor asparagine. Each of the following eight groups contains otherexemplary amino acids that are conservative substitutions for oneanother:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins        (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3rd ed., 1994) and Cantor and Schimmel, BiophysicalChemistry Part I. The Conformation of Biological Macromolecules (1980).“Primary structure” refers to the amino acid sequence of a particularpeptide. “Secondary structure” refers to locally ordered, threedimensional structures within a polypeptide. “Tertiary structure” refersto the complete three dimensional structure of a polypeptide monomer.Domains are portions of a polypeptide that form a compact unit of thepolypeptide and are typically 50 to 350 amino acids long. Typicaldomains are made up of sections of lesser organization such as stretchesof β-sheet and α-helices. Quaternary structure” refers to the threedimensional structure formed by the noncovalent association ofindependent tertiary units.

The terms “isolated” or “substantially purified,” when applied to anucleic acid or protein, denotes that the nucleic acid or protein isessentially free of other cellular components with which it isassociated in the natural state. It is preferably in a homogeneousstate, although it can be in either a dry or aqueous solution. Purityand homogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein which is the predominantspecies present in a preparation is substantially purified.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region (e.g., amino acid sequence SEQ ID NO:1 or 2), whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local alignment algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the globalalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).The Smith & Waterman alignment with the default parameters are oftenused when comparing sequences as described herein.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403410(1990), respectively. BLAST and BLAST 2.0 are used, typically with thedefault parameters, to determine percent sequence identity for thenucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid (protein) sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff& Henikoff(1989) Proc. Natl.Acad. Sci. USA 89:10915)). For the purposes of this invention, theBLAST2.0 algorithm is used with the default parameters.

A “phage display library” refers to a “library” of bacteriophages onwhose surface is expressed exogenous peptides or proteins. The foreignpeptides or polypeptides are displayed on the phage capsid outersurface. The foreign peptide can be displayed as recombinant fusionproteins incorporated as part of a phage coat protein, as recombinantfusion proteins that are not normally phage coat proteins, but which areable to become incorporated into the capsid outer surface, or asproteins or peptides that become linked, covalently or not, to suchproteins. This is accomplished by inserting an exogenous nucleic acidsequence into a nucleic acid that can be packaged into phage particles.Such exogenous nucleic acid sequences may be inserted, for example, intothe coding sequence of a phage coat protein gene. If the foreignsequence is cloned in frame, the protein it encodes will be expressed aspart of the coat protein. Thus, libraries of nucleic acid sequences,such as that of an antibody repertoires made from the gene segmentsencoding the entire B cell repertoire of one or more individuals, can beso inserted into phages to create “phage libraries.” As peptides andproteins representative of those encoded for by the nucleic acid libraryare displayed by the phage, a “peptide-display library” is generated.While a variety of bacteriophages are used in such libraryconstructions, typically, filamentous phage are used (Dunn (1996) Curr.Opin. Biotechnol. 7:547-553). See, e.g., description of phage displaylibraries, below.

Production of Antibody Sequences

Antibodies of the present invention, e.g, V_(H) polypeptides, V_(L)polypeptides, or single chain antibodies, e.g., humanized RFB4, may begenerated using routine techniques in the field of recombinant genetics.Basic texts disclosing the general methods used in this inventioninclude Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3ded. 2001) and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1999).

Humanized antibodies of the invention are generated by grafting thespecificity, i.e., the antigen binding loops, of a donor antibody,typically a murine antibody, to a human framework. The framework regionsof the human light chain and heavy chains provided herein can readily bedetermined by the practitioner. The position numbers of the heavy andlight chains are designated in accordance with common numbering schemes,e.g., the Kabat and Chothia numbering scheme. The Chothia number schemeis identical to the Kabat scheme, but places the insertions in CDR-L1and CDR-H1 at structurally different positions. Unless otherwiseindicated, the Kabat numbering scheme is used herein in reference to thesequence positions. The position of an amino acid residue in aparticular V_(H) or V_(L) sequence does not refer to the number of aminoacids in a particular sequence, but rather refers to the position asdesignated with reference to a numbering scheme.

The positions of the CDR's and hence the positions of the frameworkregions of the human heavy chain and light chains are determined usingdefinitions that are standard in the field. For example, the followingfour definitions are commonly used. The Kabat definition is based onsequence variability and is the most commonly used. The Chothiadefinition is based on the location of the structural loop regions. TheAbM definition is a compromise between the two used by OxfordMolecular's AbM antibody modelling software. The contact definition hasbeen recently introduced and is based on an analysis of the availablecomplex crystal structures. The following are the loop positions, i.e.,CDRs, using the four different definitions. Loon Kabat AbM ChothiaContact L1 L24-L34 L24-L34 L24-L34 L30-L36 L2 L50-L56 L50-L56 L50-L56L46-L55 L3 L89-L97 L89-L97 L89-L97 L89-L96 H1 H31-H35B H26-H35B H26-H32. . . 34 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35(Chothia Numbering) H2 H50-H65 H50-H58 H52-H56 H47-H58 H3 H95-H102H95-H102 H95-H102 H93-H101

A V_(H) or V_(L) sequence of the invention comprises a heavy or lightchain having a framework that typically has at least 90% identity, moretypically 95%, 96%, 97%, 98%, or 99% identity to a framework (definedusing any of the above definitions) comprised by SEQ ID NOs:1-15. Theframeworks are those residues that are not loop regions. For example,the framework of a light chain of the invention typically comprisesresidues 1-23, 35-49, 57-88, and 98-109 (or 98 through the C-terminalresidue, e.g., 98-108) using Kabat numbering. As appreciated by one ofskill in the art, these numbers may not refer to the number of aminoacids in the V_(H) or V_(L) sequence, but the positions of the residuesusing the Kabat numbering system (or other numbering system). A V_(H) orV_(L) framework sequence of the invention can thus be determined bydesignating the amino acid positions of a candidate V_(H) or V_(L)sequence using the Kabat numbering system and determining the percentidentity of the framework regions (defined using a definition providedabove) to the framework regions of a reference sequence, e.g., one ofSEQ ID NOs:1-15, which positions are designated in accordance with Kabatnumbering. The percent identity is determined to include all of theframework regions of the candidate heavy or light chain.

A humanized antibody of the invention binds to the same epitope as thedonor anti-body, e.g., a humanized RFB4 disclosed herein binds to thesame CD22 epitope, or competes for binding to the same CD22 epitope,that RFB4 binds to. Methods to determine whether the antibody binds tothe same epitope are well known in the art, see, e.g., Harlow & Lane,Using Antibodies, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1999, which discloses techniques to epitope mapping oralternatively, competition experiments, to determine whether an antibodybinds to the same epitope as the donor antibody.

A stable humanized antibody of the invention may exhibit alteredaffinity when compared to the donor antibody. For example, in someembodiments, the affinity of a single chain humanized RFB4 for CD22,may, for example, be decreased compared to a single chain antibodycomprising the RFB4 V_(H) and V_(L) regions. Such a decrease may be byas much as 10-fold in comparison, but typically a humanized antibody ofthe invention has an affinity that is at least 25%, more often at least50% of that of the comparable wildtype antibody. (A “comparable wildtypeantibody” refers to an antibody of the same embodiment, e.g., scFv, thatcomprises the donor antibody V_(H) and V_(L) regions). In someembodiments, the affinity for the epitope is increased, such that ahumanized antibody of the invention has an affinity that is 2 times andsometime 5, 10, 50, or 100 times the affinity of the comparable wildtypeantibody. Affinity may be tested as set forth in the examples.

The heavy and light chain regions of the invention are typicallyobtained using recombinant DNA technology. The recombinant DNAmethodologies that are commonly employed to perform this are well knownto those of skill in the art. Typically, nucleic acid sequences encodingthe frameworks and CDRs of the donor antibodies are generated by PCR,for example by overlap extension. In this technique, the antigen bindingsequences of the donor antibody are typically joined to the humanframework regions by incorporating the desired sequences intooligonucleotides and creating a series of products using PCR thatcomprise the desired donor and human sequences. The products may then bejoined, typically using additional PCR reactions, in the properorientation to create the V_(H) and V_(L) chains that comprise humanframework regions with donor antibody CDRs. The V_(L) and V_(H) DNAsequences may be ligated together, either directly or through a DNAsequence encoding a peptide linker, using techniques well known to thoseof skill in the art. These techniques include PCR as well as techniquessuch as in vitro ligation. The V_(L) and V_(H) sequences may be linkedin either orientation.

Mutations introduced into the framework regions, typically backmutations to the donor amino acid residue that occurs at that position,can be introduced using a number of methods known in the art. Theseinclude site-directed mutagenesis strategies such as overlap extensionPCR (see, e.g., Sambrook & Russell, supra; Ausubel et al., supra).Exemplary techniques are provided in Examples 2 and 3.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, Sambrook, and Ausubel,as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR ProtocolsA Guide to Methods and Applications (Innis et al., eds) Academic PressInc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (October 1,1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh etal. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990)Proc. Natl. Acad. Sci. USA 87, 1874; Lomeli et al. (1989) J. Clin.Chem., 35: 1826; Landegren et al., (1988) Science 241: 1077-1080; VanBrunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4:560; and Barringer et al. (1990) Gene 89: 117.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., by sequencing.

PCR products are subcloned into suitable cloning vectors that are wellknown to those of skill in the art and commercially available. Thenucleotide sequence of the heavy or light chain coding regions is thendetermined.

One of skill will appreciate that, utilizing the sequence informationprovided for the variable regions, nucleic acids encoding thesesequences are obtained using any number of additional methods well knownto those of skill in the art. Thus, DNA encoding the Fv regions isprepared by any suitable method, including, for example, otheramplification techniques such as ligase chain reaction (LCR) (see, Wu &Wallace, (1989) Genomics 4:560, Landegren, et al., (1988) Science241:1077, and Barringer, et al., (1990) Gene 89:117), transcriptionamplification (see ,Kwoh, et al., (1989) Proc. Natl Acad. Sci. USA86:1173), and self-sustained sequence replication (see, Guatelli, etal., (1990) Proc. Natl Acad. Sci. USA 87:1874), or cloning andrestriction of appropriate sequences.

The nucleic acids encoding the antibodies and antibody fragments of theinvention can also be generated by direct chemical synthesis usingmethods such as the phosphotriester method of Narang, et al., (1979)Meth. Enzymol. 68:90; the phosphodiester method of Brown, et al., (1979)Meth. Enzymol. 68:109; the diethylphosphoramidite method of Beaucage, etal., (1981) Tetra. Lett. 22:1859; and the solid support method of U.S.Pat. No. 4,458,066. If the DNA sequence is synthesized chemically, asingle stranded oligonucleotide will result. This may be converted intodouble stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. While it is possible to chemically synthesize an entire singlechain Fv region, it is preferable to synthesize a number of shortersequences (about 100 to 150 bases) that are typically later splicedtogether, for example using overlap extension PCR.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Protein sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

The V_(H) and V_(L) domains of an antibody of the invention may bedirectly linked or may be separated by a linker, e.g. to stabilize thevariable antibody domains of the light chain and heavy chain,respectively. Suitable linkers are well known to those of skill in theart and include the well known GlyGlyGlyGlySer linker or a variantthereof. For example, a typical linker is (Gly₄Ser)₃. Other linkers,including hinge regions, that can be used in the invention include thosedescribed, for example in Alfthan et al, Protein Eng. 8(7), 725-31; Choiet al, Eur. J Immunol. 31(1), 94-106; Hu et al, Cancer Res. 56(13),3055-61; Kipriyanov, et al, Protein Eng. 10(4), 445-53; Pack, et al,Biotechnology (N Y) 11(11), 1271-7; and Roovers, et al, Cancer Immunol.Immunother. 50(l):51-9.

Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding the humanized antibodies, e.g., a humanized RFB4scFv, of the invention, or an immunoconjugates comprising a humanizedantibody of the invention, one typically subclones a nucleic acidencoding the antibody or immunoconjugate into an expression vector thatcontains an appropriate promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systemsfor expressing protein are available in, e.g., E. coli, Bacillus sp.,and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al.,Nature 302:543-545 (1983). Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available.

Often, in order to express a protein at high levels in a cell, codonpreference for the expression system is considered in constructing thenucleic acid sequence to be expressed. Thus, a nucleic acid from oneorganism, e.g., a human or mouse, may be engineered to accommodate thecodon preference of the expression system.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the protein-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding theprotein to be expressed and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. The nucleic acid sequence encoding a proteinmay typically be linked to a cleavable signal peptide sequence topromote secretion of the encoded protein by the transformed cell. Suchsignal peptides would include, among others, the signal peptides fromtissue plasminogen activator, insulin, and neuron growth factor, andjuvenile hormone esterase of Heliothis virescens. Additional elements ofthe cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21-25); and the lambda-derived P_(L) promoter andN-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128).The particular promoter system is not critical to the invention, anyavailable promoter that functions in prokaryotes can be used.

Standard bacterial expression vectors include plasmids such aspBR322-based plasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, λ-phage derivedvectors, and fusion expression systems such as GST and LacZ. Epitopetags can also be added to recombinant proteins to provide convenientmethods of isolation, e.g., c-myc, HA-tag, 6-His tag, maltose bindingprotein, VSV-G tag, anti-DYKDDDDK tag, or any such tag, a large numberof which are well known to those of skill in the art.

Eukaryotic expression systems for mammalian cells, yeast, and insectcells are well known in the art and are also commercially available. Inyeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and YeastReplicating plasmids (the YRp series plasmids) and pGPD-2. Expressionvectors containing regulatory elements from eukaryotic viruses aretypically used in eukaryotic expression vectors, e.g., SV40 vectors,papilloma virus vectors, and vectors derived from Epstein-Barr virus.Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the CMV promoter, SV40 earlypromoter, SV40 later promoter, metallothionein promoter, murine mammarytumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter,or other promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a GPCR-encoding sequence underthe direction of the polyhedrin promoter or other strong baculoviruspromoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressing apolypeptide of the invention.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe protein, which is recovered from the culture using standardtechniques identified below.

One of skill would recognize that modifications can be made to a nucleicacid encoding a polypeptide of the present invention (i.e., an antibody,a label or effector, or an immunoconjugate formed using the antibody)without diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, terminationcodons, a methionine added at the amino terminus to provide aninitiation, site, additional amino acids placed on either terminus tocreate conveniently located restriction sites, or additional amino acids(such as poly His) to aid in purification steps.

Once expressed, the recombinant antibodies, immunoconjugates, and/oreffector molecules of the present invention can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N.Y.(1982)). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, if to be used therapeutically, the polypeptidesshould be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies of this invention. See, Buchner, et al.,Anal Biochem. 205:263-270 (1992); Pluckthun, Biotechnology 9:545 (1991);Huse, et al., Science 246:1275 (1989) and Ward, et al., Nature 341:544(1989), all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well-known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena, et al., Biochemistry 9: 5015-5021 (1970),incorporated by reference herein, and especially as described byBuchner, et al., supra.

Renaturation is typically accomplished by dilution (e.g., 100-fold) ofthe denatured and reduced protein into refolding buffer. An exemplarybuffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. A preferred yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. It isdesirable to add excess oxidized glutathione or other oxidizing lowmolecular weight compounds to the refolding solution after theredox-shuffling is completed.

In addition to recombinant methods, the antibodies and immunoconjugatesof the invention can also be constructed in whole or in part usingstandard peptide synthesis. Solid phase synthesis of the polypeptides ofthe present invention of less than about 50 amino acids in length may beaccomplished by attaching the C-terminal amino acid of the sequence toan insoluble support followed by sequential addition of the remainingamino acids in the sequence. Techniques for solid phase synthesis aredescribed by Barany & Merrifield, THE PEPTIDES: ANALYSIS, SYNTHESIS,BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS, PART A. pp.3-284; Merrifield, et al. J. Am. Chem. Soc. 85:2149-2156 (1963), andStewart, et al., SOLID PHASE PEPTIDE SYNTHESIS, 2ND ED., Pierce Chem.Co., Rockford, Ill. (1984). Proteins of greater length may besynthesized by condensation of the amino and carboxyl termini of shorterfragments. Methods of forming peptide bonds by activation of a carboxylterminal end (e.g., by the use of the coupling reagent N,N′-dicycylohexylcarbodiimide) are known to those of skill.

Conservatively Modified Variants

Conservatively modified variants of have at least 80% sequencesimilarity, often at least 85% sequence similarity, 90% sequencesimilarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarityat the amino acid level, with the protein of interest, such as ahumanized RFB4 of the invention.

As noted in the “definitions” section, the term “conservatively modifiedvariants” can be applied to both amino acid and nucleic acid sequences.With respect to particular nucleic acid sequences, conservativelymodified variants refer to those nucleic acid sequences which encodeidentical or essentially identical amino acid sequences, or if thenucleic acid does not encode an amino acid sequence, to essentiallyidentical nucleic acid sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode any given polypeptide. For instance, the codons GCA, GCC, GCG andGCU all encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Such substitutions may be at a position defined herein that isback-mutated, or at another position.

For example, SGIII has a leucine substituted for the arginine thatoccurs at that position in the human V_(L)#19 sequence. A valine mayalso be substituted for the arginine, which substitution should noteffect the binding affinity or stability. The effects of suchsubstitutions can readily be determined by measuring binding and/oraffinity using the methodology described herein. Conservative changed toV_(H) and V_(L) domains may also be introduced at other positions, e.g.,in the core region. The effects of the substitution may readily bedemonstrated by determining effects on stability and/or affinity asdescribed herein.

Immunoconjugates

One embodiment of the present invention provides an immunoconjugatecomprising a humanized antibody of the invention, e.g., a humanized RFB4antibody, linked to an effector molecule or detectable label. Preferablythe effector molecule is a therapeutic molecule such as, for example, atoxin, a small molecule, a cytokine or a chemokine, an enzyme, or aradiolabel. Exemplary toxins include, but are not limited to,Pseudomonas exotoxin or diphtheria toxin. Suitable toxins are describedin e.g., Chaudhary, et al. (1987) Proc Natl Acad Sci US A 84:4538,Chaudhary, et al. (1989) Nature 339:394, Batra, et al. (1991) Mol CellBiol 11:2200. Brinkmann, et al. (1991) Proc Natl Acad Sci USA 88:8616,Siegall, (1995) Semin Cancer Biol 6:289. Examples of small moleculesinclude, but are not limited to, chemotherapeutic compounds such astaxol, doxorubicin, etoposide, and bleiomycin. Exemplary cytokinesinclude, but are not limited to, IL-1, IL-2, IL-4, IL-5, IL-6, andIL-12. Suitable cytokines and chemokines are described in, e.g.,Rosenblum et al. (2000) Int J Cancer 88:267 and Xu et al. (2000) CancerRes 60:4475 and Biragyn et al. (1999) Nat Biotechnol 17:253. Exemplaryenzymes include, but are not limited to, RNAses, DNAses, proteases,kinases, and caspases. Suitable proteases are described in, e.g.,Bosslet et al. (1992) Br J Cancer 65:234, Goshom et al. (1993) CancerRes 53:2123, Rodrigues et al. (1995) Cancer Res 55:63, Michael et al.(1996) Immunotechnology 2:47, Haisma et al. (1998) Blood 92:184.Exemplary radioisotopes include, but are not limited to, ³²P and ¹²⁵I.Suitable radionuclides are also described in, e.g., Colcher et al.(1999) Ann N Y Acad Sci 880:263. Additional exemplary effector moietiesare, for example, Fc fragments from homologous or heterologousantibodies.

In some embodiments, RNase A family members are conjugated to ahumanized antibody of the invention. Exemplary RNAses include RapLR1,and angiogenin. Suitable RNAses are also described in Newton et al.(1994) J Biol Chem 269:26739, Newton, et al. (1996) Biochemistry 35:545,and Zewe, et al. (1997) Immunotechnology 3:127-136. RapLR1 variants canbe generated that have essentially the same activity as a native RapLR1.Variants of recombinant RNases, and techniques for synthesizing theseproteins, are described in PCT/US97/02588 and WO99/50398.

It will be appreciated by those of skill in the art that the sequence ofany protein effector molecule may be altered in a manner that does notsubstantially affect the functional advantages of the effector protein.For example, glycine and alanine are typically considered to beinterchangeable as are aspartic acid and glutamic acid and asparagineand glutamine. One of skill in the art will recognize that manydifferent variations of effector sequences will encode effectors withroughly the same activity as the native effector.

The effector molecule and the antibody may be conjugated by chemical orby recombinant means as described above. Chemical modifications include,for example, derivitization for the purpose of linking the effectormolecule and the antibody to each other, either directly or through alinking compound, by methods that are well known in the art of proteinchemistry. Both covalent and noncovalent attachment means may be usedwith the humanized antibodies of the present invention.

The procedure for attaching an effector molecule to an antibody willvary according to the chemical structure of the moiety to be attached tothe antibody. Polypeptides typically contain a variety of functionalgroups; e.g., carboxylic acid (COOH), free amine (—NH₂) or sulfhydryl(—SH) groups, which are available for reaction with a suitablefunctional group on an antibody to result in the binding of the effectormolecule.

Alternatively, the antibody is derivatized to expose or to attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford Ill.

The linker is capable of forming covalent bonds to both the antibody andto the effector molecule. Suitable linkers are well known to those ofskill in the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (e.g., through a disulfide linkage to cysteine). However, ina preferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

In the presently preferred chemical conjugation embodiment, the means oflinking the effector molecule and the antibody comprises aheterobifunctional coupling reagent which ultimately contributes toformation of an intermolecular disulfide bond between the effectormolecule and the antibody. Other types of coupling reagents that areuseful in this capacity for the present invention are described, forexample, in U.S. Pat. No. 4,545,985. Alternatively, an intermoleculardisulfide may conveniently be formed between cysteines in the effectormolecule and the antibody which occur naturally or are inserted bygenetic engineering. The means of linking the effector molecule and theantibody may also use thioether linkages between heterobifunctionalcrosslinking reagents or specific low pH cleavable crosslinkers orspecific protease cleavable linkers or other cleavable or noncleavablechemical linkages. The means of linking the effector molecule and theantibody may also comprise a peptidyl bond formed between the effectormolecule and the antibody which are separately synthesized by standardpeptide synthesis chemistry or recombinant means.

Exemplary chemical modifications of the effector molecule and theantibody of the present invention also include derivitization withpolyethylene glycol (PEG) to extend time of residence in the circulatorysystem and reduce immunogenicity, according to well known methods (Seefor example, Lisi, et al., Applied Biochem. 4:19 (1982); Beauchamp, etal., Anal Biochem. 131:25 (1982); and Goodson, et al., Bio/Technology8:343 (1990)).

Antibodies of the present invention may optionally be covalently ornon-covalently linked to a detectable label. Detectable labels suitablefor such use include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. DYNABEADS), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

Pharmaceutical Compositions And Administration

The antibody and/or immunoconjugate compositions of this invention areparticularly useful for parenteral administration, such as intravenousadministration or administration into a body cavity.

The compositions for administration will commonly comprise a solution ofthe antibody and/or immunoconjugate dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of fusion protein in these formulations can vary widely,and will be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs.

Thus, a typical pharmaceutical immunotoxin composition of the presentinvention for intravenous administration would be about 0.1 to 10 mg perpatient per day. Dosages from 0.1 up to about 100 mg per patient per daymay be used. Actual methods for preparing administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as REMINGTON'S PHARMACEUTICALSCIENCE, 19TH ED., Mack Publishing Company, Easton, Pa. (1995).

The compositions of the present invention can be administered fortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. An effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the proteins of this invention to effectively treat thepatient. Preferably, the dosage is administered once but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the patient.

Controlled release parenteral formulations of the immunoconjugatecompositions of the present invention can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS:FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μm so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly. See,e.g., Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed.,Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice &Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., MarcelDekker, Inc. New York, N.Y., pp.315-339, (1992) both of which areincorporated herein by reference.

Polymers can be used for ion-controlled release of immunoconjugatecompositions of the present invention. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)).For example, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston, etal., Pharm. Res. 9:425-434 (1992); and Pec, et al., J. Parent. Sci.Tech. 44(2):58-65 (1990)). Alternatively, hydroxyapatite has been usedas a microcarrier for controlled release of proteins (Ijntema, et al.,Int. J. Pharm. 112:215-224 (1994)). In yet another aspect, liposomes areused for controlled release as well as drug targeting of thelipid-capsulated drug (Betageri, et al., LIPOSOME DRUG DELIVERY SYSTEMS,Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerousadditional systems for controlled delivery of therapeutic proteins areknown. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871,4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670;5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961;5,254,342 and 5,534,496, each of which is incorporated herein byreference.

Among various uses of the immunoconjugates of the invention are includeda variety of disease conditions caused by specific human cells that maybe eliminated by the toxic action of the fusion protein. For example,for the humanazied RFB4 antibodies disclosed herein, one preferredapplication for immunoconjugates is the treatment of malignant cellsexpressing CD22. Exemplary malignant cells include those of chroniclymphocytic leukemia and hairy cell leukemia.

Diagnostic Kits And Uses

In another embodiment, this invention provides kits for the detection ofantigens, e.g., CD22, or an immunoreactive fragment thereof, in abiological sample. A “biological sample” as used herein is a sample ofbiological tissue or fluid that contains the antigen. Such samplesinclude, but are not limited to, tissue from biopsy, blood, and bloodcells (e.g., white cells). Preferably, the cells are lymphocytes.Biological samples also include sections of tissues, such as frozensections taken for histological purposes. A biological sample istypically obtained from a multicellular eukaryote, preferably a mammalsuch as rat, mouse, cow, dog, guinea pig, or rabbit, and more preferablya primate, such as a macaque, chimpanzee, or human. Most preferably, thesample is from a human.

The antibodies of the invention may also be used in vivo, for example,as a diagnostic tool for in vivo imaging.

Kits will typically comprise an antibody of the current invention. Insome embodiments, the antibody will be a humanized anti-CD22 Fvfragment, such as a scFv or dsFv fragment.

In addition the kits will typically include instructional materialsdisclosing means of use of an antibody of the present invention (e.g.for detection of mesothelial cells in a sample). The kits may alsoinclude additional components to facilitate the particular applicationfor which the kit is designed. Thus, for example, the kit mayadditionally contain means of detecting the label (e.g. enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a sheep anti-mouse-HRP, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

In another set of uses for the invention, immunotoxins targeted byantibodies of the invention can be used to purge targeted cells from apopulation of cells in a culture. Thus, for example, cells cultured froma patient having a cancer expressing CD22 can be purged of cancer cellsby contacting the culture with immunotoxins which use the antibodies ofthe invention as a targeting moiety.

Specificity Grafting Using New Human Frameworks of Unknown ThreeDimensional Structure

The invention also provides methods for identifying stable humanframeworks that can be used as acceptors for donor bindingspecificities. Such sequences can be obtained after stringent panning ofa human display library with an antigen. The stringent panning procedureresults in selection of stable, soluble scFv antibodies (see, e.g.,Hoogenboom & Winter, J. Mol. Biol. 224:381, 1992; Sheets et al., Proc.Natal. Acad. Sci. USA 95:6157, 1998; Visintin, et al., J. Mol. Biol.317:73, 2002); Jung and Pluckthun, Protein Eng. 10:959, 1997; Chowdhuryet al., Proc. Natl. Acad. Sci USA 95:669, 1998). These antibody variabledomain frameworks are subsequently used as human acceptor scaffold forgrafting the murine antibody specificity. The method described hereindiffers from other humanization procesures in which human acceptorscaffold are selected from either antibodies with solved crytalstructure or (germline) sequence databases. Typically, in the currentinvention, human acceptor frameworks are first pre-selected forstability by screening display libraries under stringent conditions.Framework sequence with high sequence identities to the murine antibodyto be humanized are then chosen from the pre-selected pool of stablescaffolds. As a result, humanized scFv with low immunogenic potentialand high biophysical stabilitiy are generated.

Human framework sequences can be obtained by the skilled artisan usingwell known techniques, e.g., using phage display libraries (see, e.g.,Sastry et al, Proc Natl Acad Sci USA 86:5728-5732, 1989; McCafferty etal., Nature 348:552-554, 1990; Marks et al, J Mol Biol 222:581-597,1991; Clackson et al, Nature 352:624-628, 1991; and Barbas et al, ProcAcad Sci USA 88:7978-7982, 1991) to isolate stable human V_(H) and V_(L)sequences, for example, corresponding to the B-cell repertoire of one ormore individuals. The sequences are determined using standardtechnology.

A pre-selected pool of stable scaffols is selected as follows. A humandisplay library, e.g., a phage display library that expresses humanV_(H) and/or V_(L) sequences, is screened with a screening antigen understringent conditions as set forth in Schmidt et al, supra. The selectionprotocol comprises at least two panning rounds, typically three, andmore often four or more. The screening antigen can be any antigen, e.g.,lysozyme as the end result to be achieved at this step is to identifystable frameworks, not an antibody with a particular bindingspecificity. The selection procedure results in a pool of clones thatare enriched for stable, soluble antibody sequences. This pool is usedas a source for human framework sequences that can be used as acceptorsfor donor CDR sequences that are from an antibody that has the bindingspecificity of interest.

V_(H) and V_(L) amino acid sequences from the pre-selected pool ofclones are then aligned with a donor antibody of interest to selectframeworks for humanizing the donor antibody. In brief, the heavy andlight chain variable sequences of a donor antibody of interest, e.g., amurine monoclonal antibody, are aligned with the cloned human heavy andlight chain sequences. The human sequences that have sequence identityof at least about 70% or greater are selected for candidate frameworksequences for humanization.

In some embodiments, e.g., in instances in which for murine frameworksand human acceptor scaffolds that are closely related and in which themurine structural loops from canonical structures, the invention furtherprovides methods of identifying framework residues that are importantcandidates for back-mutation in humanization studies. A general protocolis provided below. The steps of the protocol are presented in apreferable order with the understanding that one of skill in the art mayalter some of the procedures.

1. Preferably, the antibody subgroups of the acceptor and donorsequences are determined. The antibody subgroup of the (typicallydetermined according to KABAT) of the human acceptor variable lightchain and variable heavy chain candidates are determined by aligning therespective amino acid sequences to the publicly available KABATdatabase, Kabat Database of Sequences of Proteins of ImmunologicalInterest, Feb. 22, 2002 Dataset)(http://immuno.bme.nwu.edu/subgroup.html). The donor antibody variablelight and heavy chain sequences are also aligned to human subgroups andthe closest human subgroups to these sequences identified. An overallidentity (>60%) is typically expected.

2. The numbering of the residues in the light and heavy chain variableregions are typically determined according to KABAT, e.g., using theAbcheck software, e.g. available at http://www.bioinf.org.uk/abs/ (e.g.,Martin, A. C. R. (1996) Accessing the Kabat antibody sequence databaseby computer. PROTEINS: Structure, Function and Genetics, 25, 130-133).The software aligns the provided sequence to a consensus sequence to mapit to the Kabat numbering system. In an additional step, the alignedsequence is scanned against the Kabat database (currently containingsequences of 2707 light chains and 3471 heavy chains) to identifyresidues that are “unusual”, i.e., they occur in <1% of the sequences.

3. Antigen binding loop regions may be identified using a number ofantigen binding loop definitions such as those by Kabat, Chothia, IMGT(Ruiz, et al., Nucleic Acids Res. 28:219-221 (2000); and Lefranc, Nucl.Acids Res. 29:207-9 (2001)), AbM (Martin et al., Proc. Natl Acad. Sci.USA, 86:9268-9272, (1989); Martin et al, Methods Enzymol. 203:121-153(1991); Pedersen et al, Immunomethods 1:126 (1992); and Rees et al, InSternberg M.J.E. (ed.), Protein Structure Prediction. Oxford UniversityPress, Oxford, 141-172, (1996)), and contact (MacCallum et al., J ofMol. Biol. 262:732-745 (1996)).

4. Acceptor and donor amino acid sequences are aligned to each other.Typically, those frameworks that exhibit at least about 70% identity areselected.

5. Potential N- or O-glycosylation sites are also typically identified,e.g., by scanning sequence against Expasy tools server athttp://www.cbs.dtu.dk/services/NetOGlyc/ (Cooper et al, GlycoMod—asoftware tool for determining glycosylation compositions from massspectrometric data Proteomics 2001 Feb; 1(2):340-9)

6. Donor and acceptor sequences are aligned with reference sequences,e.g., those discussed below, to identify framework residues that arecandidates for backmutation to the donor sequence. Candidate residuesfor backmutation include:

Residues that are “invariant” according to previously identified humansubgroups (=residues present in greater than about 95% of all entries inKabat database) according to Kabat et al., February 2002, Sequences ofimmunological interest. US Department of Health and Human Services).

“Key residues” that determine the main chain conformations of theantigen binding loops L1-L3, H1, H2 may be found at the followingpositions (see, e.g., Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917;Chothia. et al., 1989, Nature 342, 877-883; Chothia et al., 1992, J.Mol. Biol. 227, 799-817): L1 residues 2, 25, 29, 33, 71 L2 residues 48,64 L3 90, 95 H1 24, 26, 27, 29, 34, 94 H2 52a, 54, 55, 71 H3 structuretoo complex, key residues not yet identified

Main chain conformations of antigen binding loops can be subdivided intodifferent canonical classes. One or more canonical classes are known forloops L1-3, H2, H3. The canonical class assignments of donor L1-L3, H1,H2 loops are determined using methods known in the art.

The residues at the V_(H)/V_(L) interface, which are known in the art(see, e.g., http://www.cryst.bbk.ac.uk/˜ubcg07s/; Novotny et al,,Molecular anatomy of the antibody binding site. J Biol Chem 1983 Dec10;258(23):14433-7; Novotny & Haber, Structural invariants of antigenbinding: comparison of immunoglobulin VL-VH and VL-VL domain dimers,Proc Natl Acad Sci USA 1985 Jul;82(14):4592-6; Chothia et al, Domainassociation in immunoglobulin molecules. The packing of variabledomains. J Mol Biol 1985 December 5;186(3):651-63).

7. Mismatches in the key residues (as defined by Chothia) between donorand acceptor sequence are identified through application of Kabatnumbering to the candidate and donor sequences and comparison of thesequences. (Mismatches within the predefined CDR's are not considered.)

8. Mismatching key residues within the framework regions are all changedto donor sequences.

9. The significance of the identified mismatching residues through thedescribed sequence multialignment is determined on the basis ofinformation of other antibody variable domains with similar structures.

10. A hierarchy from most important to least important mismatchedframework residues may be determined to assist in creating humanizedantibodies that maintain binding properties of the donor antibody. Forexample, the hierarchy may be determined as follows. First, thosemismatched residues that are of known importance, for example H6, aretypically include in the first tier of such a hierarchy, i.e.,mismatches in these residues may be initial residues that arebackmutated. Then residues that may have profound consequences onframework binding properties, for example key residues, may be includesin a second tier. A third tier may, for example, include potential keyresidues, such as those described in Martin & Thomton, J. Mol. Biol.263:800-815, 1996. Lastly, secondary key residues may be considered. Theresidues included at this level are typically those that are unusual atthat position.

The donor specificity is then grafted to the human framework sequenceusing well known techniques, e.g., PCR, to generate a humanizedantibody. Variants of the humanized antibody that have desirable bindingproerties may be generated, typically based on the hierarchy. Often, thevariants are sequences that have the particular residues identified bythe hierarchy backmutated to the donor sequence. Other variants may alsocomprise conservative substitutions at positions that do not effect thestability of the humanized antibody.

As appreciated by one of skill in the art, this method can be applied toany framework region for which the sequence is determined. Variations onthe steps of the method are also possible, for example, the selection ofan optimal numbering scheme, which variations do not change the method.

EXAMPLES Example 1

Preparation of Phase Display Library from Human B-cells

A scFv antibody phage display library with a repertoire of 5x 106individual clones was generated from lymph node biopsies of two patientswith B-cell non-Hodgkin lymphoma as described (Mao et al., Proc NatlAcad Sci USA 96:6953-6958, 1999). To generate the libraries, antibodyvariable gene segments of the entire B cell repertoire of these patientswere amplified by PCR and cloned into a phagemid vector. The variableregions were thus expressed as randomly associated variable domainfragments on the surface of filamentous phage. This library wassubjected to four rounds of in vitro panning on Daudi tumor cells asdescribed (Schmidt et al., Biotechniques 26:697-702, 1999). A pool ofenriched scFv antibodies was obtained and several phage antibodies thathad good production characteristics, that is produced >1 mg/l bacterialculture, were identified and sequenced. The sequences of some of theseclones are provided in the Table of Sequences. The V_(H) and V_(L)sequences were then evaluated as potential acceptor frameworks forspecificity grafting.

Variable domain framework sequences of the clones were aligned tocorresponding sequences of the murine monoclonal antibody RFB4. A subsetof four V_(H) (all human V_(H)III subgroup) and four V_(L) (all humanVκ1 subgroup) human scaffolds each showed ≧70% sequence identity tocorresponding sequences of the murine mAb RFB4 (Mansfield et al, supra).These human frameworks were screened against V-Base to identify theirclosest corresponding human germline sequences. J genes were analyzedseparately.

Aligning the best matching human FR-V_(L) clone #19(73.8% sequenceidentity to murine FR-V_(L)-RFB4) to the closest human variable domaingermline sequence (HSIGKLA30, Vκ1-17) also revealed the lowest number ofsomatic framework mutations when compared with the other pre-selectedlight chains. Separate alignment of the human J-gene segment showed acomplete match to human Jκ2 (FIG. 2).

Similarly, human clone V_(H#)8 with the highest sequence identity tomurine FR-V_(H)-RFB4 (81.7%) also showed, in comparison with the otherV_(H) candidates, the best match to the closest human V gene germlinesegment (HSIGVH81B, VH3-3-66). Three non-identical residues withinCDR-H3 of the rearranged human JH4 gene segment were identified (FIG.2). Thus frameworks of VL#19 and VH#8 showed the best match to both themurine mAb to be humanized and corresponding human germline sequencesand were therefore selected for specificity grafting.

A general strategy was employed to identify unusual framework residuesin the human sequence that may influence the structural integrity of theantigen binding site and to back-mutate these to murines residues,particularly if this restored the human germline residue at the sametime. To identify such residues, both murine donor and selected humanacceptor sequences were aligned to several sequence templates derivedfrom antibody repertoires. With the exception of CDR-H1, the antigenbinding loops to be grafted onto the human framework regions weredefined according to Kabat et al. (1991) Sequences of Proteins ofImmunological Interest. (NIH Publication No. 91-3242, Bethesda). Asresidues H26-H32 comprise the structural loop of CDR-H1 (Chothia et al,Nature 342:877-883, 1989), residues H26-H35 were applied as CDR-H1according to the combined Kabat/Chothia definition of CDR-H1.

“Invariant residues” (Kabat et al., 1991, supra) and “key residues”(Chothia et al., 1989, supra) were identified, and canonical-classassignments of the donor antigen binding loops Ll-L3, HI and H2,respectively, were determined by screening the sequence against sequencetemplates (Martin and Thornton, J Mol Biol. 263:800-815, 1996) athttp://www.bioinf org.uk/. Furthermore, residues at the V_(H)/V_(L)interface (Chothia et al., J Mol Biol. 186:651-663, 1985) and residuesknown to be structurally conserved at core sites (Chothia et al., J MolBiol 278:457-479, 1998), were compared with corresponding donor andacceptor residues. Non-matching donor and acceptor framework residues atthese sites were analyzed on the basis of information from otherantibodies of known structure from the Protein Databank (Berman et al.,Nucleic Acids Res, 28:235-242, 2000).

The “invariant” residues were defined as those residues present in ≧95%of all entries in Kabat database.

“Key residues” were identified that determine the main chainconformations of the antigen binding loops L1-L3, H1, H2 at thefollowing positions (see, e.g., Chothia C. and Lesk A. M., 1987, J. Mol.Biol. 196, 901-917; Chothia C. et al., 1989, Nature 342, 877-883;Chothia C. et al., 1992, J. Mol. Biol. 227, 799-817): L1 residues 2, 25,29, 33, 71 L2 residues 48, 64 L3 90, 95 H1 24, 26, 27, 29, 34, 94 H252a, 54, 55, 71 H3 structure too complex, key residues not yetidentified

Using this strategy, uncommon residues (they occur in <1% of thesequences in the database) within both the V_(H) and V_(L) frameworkregions were identified (FIG. 2). The potential significance of theseresidues was analyzed on the basis of structural information of antibodyrepertoires, as described above. Nine residues were predicted to affectantigen binding and thus considered back-mutation candidates. Eachselected residue is discussed in turn.V_(L)-3E→Q

In all human sequences in Kabat, valine, glutamate, glutamine andalanine are all common at V_(L)-3. However, glutamine (Q) is the mostcommon residue of human subgroup V_(κ)1 at this position and is presentin the germline sequence corresponding to V_(L#)19. Thus, glutamate atV_(L)-3 in the acceptor sequence was likely to have been introduced bythe use of a degenerate 5′ primer and this location was thus considereda prime candidate for back-mutation to the murine donor residue which isglutamine.V_(L)-40V→P

The valine (V) at V_(L)-40 in the acceptor sequence was particularlyunusual for human or mouse sequences and did not match the closestcorresponding germline sequence. The Kabat database contains only threesequences with valine at this location whereas proline is extremelycommon. This proline is involved in a hairpin structure at the rear endof FRII (away from the combining site) which, in turn, may have aneffect on the conformation of the framework supporting CDR-L1 andCDR-L2. Hence this residue, was predicted to be very important.V_(L)-46R→L

Residue V_(L)-46 is involved in an interface contact with V_(H)-47 andalso has a minor packing role in canonical class 2 CDR-L1 (Martin andThornton, 1996, supra). These two factors suggest that it is involved indefining the conformation of CDR-L1 and will have an influence onV_(H)/V_(L) packing. A back-mutation from arginine to leucine atV_(L)-46 was therefore predicted as potentially important.V_(L)-49S→Y

The VL#19 sequence was predicted to adopt a CDR-L1 conformation similarto canonical class 2/11A as defined by Martin & Thornton (Martin andThomton, 1996, supra). However, their analysis of conformational classesrequires tyrosine, histidine, phenyl-alanine or lysine at V_(L)-49, aresidue against which CDR-L1 packs. The human VL#19 sequence has aserine at this location whereas the mouse donor sequence has tyrosine.Back-mutation of this residue was therefore considered important.V_(L)-71F→Y

Residue V_(L)-71 is also involved in packing with CDR-L1. However, bothV_(L)-71Y in the murine donor sequence and V_(L)-71F in the VL#19acceptor framework sequence allow CDR-L1 to adopt canonical class 2.This residue was predicted to have only a minor effect on theconformation of CDR-L1.V_(H)-6Q→E

Residue V_(H)-6, a major determinant of the framework H1 conformation,was previously shown to be highly critical for antigen binding (de Haardet al., Protein Eng, 11:1267-1276, 1998; Honegger and Pluckthun J MolBiol, 309, 687-699, 2001; Jung et al., J Mol Biol. 309:701-716, 2001).Mutation from glutamine to glutamate was therefore predicted to be veryimportance.V_(H)-40V→T

Hydrophobic residues at V_(H)-40 such as the valine in the acceptorsequence are only seen in 8% of rearranged and 6% of germline genes,respectively. However, since V_(H)-40 is located at the back of the Fvaway from the combining site, a back-mutation to the donor sequence waspredicted to have only a minor effect.V_(H)-79D→Y

The aspartate present in the human acceptor is unusual with the mostcommon residue at V_(H)-79 in human sequences being tyrosine, as seen inthe mouse donor. A neutral residue is present in 89% of sequences (95%of germline). While not interacting directly with the CDRs, V_(H)-79packs against the H0 loop against which the CDRs pack and may thus havean indirect effect on CDR conformation. (The H0 and L0 loops occurbetween the 5^(th) and 6^(th) strands of the immunoglobulin fold andthus lie on the same side of the antibody as the CDRs, but are nothypervariable and do not form part of the combining site.) This residueis in a location similar to V_(H)-23 which has been shown previously tohave a small effect on binding (Adair et al., Hum Antibodies Hybridomas5:41-47, 1994).V_(H)-84V→S

Position V_(H)-84 is a valine in the human acceptor. Hydrophobicresidues are seen in only 9% of human sequences (3% of germline) at thislocation. However, like V_(H)-40, residue V_(H)-84 is located at therear of the Fv and was therefore expected to have only a minor effect onbinding.

Based on this analysis, a hierarchy of the expected importance ofmutations was generated (Table I) and six specificity grafted versionswith successive donor residue back-mutations were designed accordingly(Table II). TABLE I Hierarchy of critical framework residues

TABLE II Specificity grafted variants with backmutations to murine donorsequence Yield Variant Backmutations (μg/L)^(a) K_(d) (nM)^(b) V_(H)V_(L) SG0 — — 21 —^(c) SGI Q6E E3Q 26 —^(c) SGII Q6E E3Q, V40P, S49Y 37462.6 ± 2.1  SGIII Q6E E3Q, V40P, S49Y, 42 9.8 ± 1.1 R46L SGIV Q6E, D79YE3Q, V40P, S49Y, 45 6.5 ± 0.6 R46L, F71Y SGV Q6E, D79Y, E3Q, V40P,S49Y,Y 42 9.6 ± 0.3 V40T, V84S R46L, F71^(a)Yield refers to monomeric scFv protein after purification byimmobilized metal chelate chromatography and size exclusionchromatography.^(b)Binding affinity constants (K_(d)) were determined by fitting thecell binding data from FIG. 3 to the nonlinear regression modelaccording to the Levenberg-Marquard method.^(c)K_(d) quantitatively not determinable due to fast dissociation rate.

Example 2

Generation of Humanized RFB4 scFvs

A scFv comprising the human V_(H#)8 heavy chain and the V_(L#)19 lightchain, respectively (FIG. 2) was first constructed. This construct wasproduced as soluble protein but binding was not specific to the CD22antigen (data not shown). Six specificity grafted scFv mutants weregenerated subsequently (Table II).

In order to generate an scFv with good production properties, i.e.,expression characteristics, the V_(L) and V_(H) encoding genes weresynthesized by overlap extension PCR techniques, considering anoptimized codon usage for E. coli for primer design.

CDR-grafted variant scFv SGO was generated by sequential PCRs usingeight overlapping oligonucleotides each for the construction of V_(H)and V_(L) and overlap extension techniques as described (Ye et al.,Biochem Biophys Res Commun 186:143-149, 1992). A standard (Gly₄Ser)₃linker connecting the V_(H) and V_(L) domains was likewise introduced byPCR. Silent mutations were introduced into primers such that the codonusage was adapted for optimized expression of the constructs in E. coliby eliminating the most unusual codons for prokaryotic proteinexpression Leu-CTA, Pro-CCC, Ile-ATA, Arg-AGA, Arg-AGG. PCR productsencoding scFv SGO were cleaved with appropriate restriction enzymes andligated into pHOG 21 (Kipriyanov et al., J Immunol Methods 196:51-62,1996) for soluble expression. Variants SGI-SGV were constructed by sitedirected mutagenesis and overlap extension PCR as described (Ho et al.,Gene 77:51-59, 1989) using scFv SG0-DNA as a template.

Example 3

Expression and Purification of SG scFv Variants

The E. coli strain TG1 (Stratagene, La Jolla, Calif.), transformed withthe scFv expression plasmid, was grown at 37° C. and 230 rpm in 1000 ml2YT medium containing 100 μg/ml ampicillin and 100 mM glucose (2YTGA).Cells were pelleted by centrifugation after reaching an OD600 of 0.8-1.0at 1500 g for 20 min at 20° C. and resuspended in the same volume offresh 2YT medium containing 100 μg/ml ampicillin, 0.4 M sucrose and 1 mMIPTG. Induction was performed at 19° C. for 18-20 h. Bacteria werepelleted by centrifugation at 7000 g, 30 min at 4° C., resuspended in 5%of the initial volume in periplasmic extraction buffer (50 mM Tris, 1 mMEDTA, 20% Sucrose, pH 8.0) and incubated for 1 h on ice. The suspensionwas centrifuged at 30,000 g at 4° C. for 1 h and the solublescFv-containing supernatant was thoroughly dialyzed against SP10 buffer(300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH 8.0). The dialyzedcrude periplasmic extract was purified by immobilized metal affinitychromatography (IMAC) using Ni-NTA columns according to the protocol ofthe manufacturer (Qiagen, Valencia, Calif.). Eluted, purified scFvantibodies were extensively dialyzed against PBS, 50 mM imidazole.

Monomeric scFv fragments were separated from higher molecular forms bysize-exclusion chromatography using a calibrated Superdex 75 HR 10/30column (Amersham Pharmacia, Piscataway, N.J.). Monomeric scFv fractionswere analyzed on 4-20% SDS-PAGE under reducing conditions and stainedwith Simply Blue™ Safe Stain (Invitrogen, Carlsbad, Calif.), or byWestern blot, using anti-c-myc mAb 9E10 (Roche, Indianapolis, Ind.) asfirst, and alkaline phosphatase conjugated anti-mouse IgG (Sigma, St.Louis, Mo.) as secondary, antibody. Concentrations of monomeric scFvcorresponding fractions were determined by measuring the absorbance atA280 nm with a spectrophotometer.

Monomeric protein fractions with apparent molecular masses ofapproximately 29 kDa could be well separated from a small fraction ofdimers (<8%, except variant SGII producing 28% dimeric protein) bysize-exclusion chromatography (Table II).

Example 4

Stability of the Humanized RFB4 Antibodies.

Variants were further tested for stability and affinity.

METHODS

Binding and Competition Assays

Specific binding of the constructs was determined by flow cytometryusing the human CD22⁺ B cell lines Raji, Ramos, Daudi and CA46. Human Tcell lines Jurkat and HUT102 were used as negative controls. Cells(5×10⁵) were incubated with 100 μl of a sample containing either thescFv fragments, or control antibodies, in FACS buffer (PBS, 0.1% NaN₃,2% FBS) for 45 min at 4° C. in round bottom 96-well microtiter plates.Cells were pelleted at 200 g at 4° C. for 5 min and washed twice with200 μl FACS buffer. For detection of bound antibodies, cells were firstincubated for 30 min at 4° C. with saturating concentrations of theanti-c-myc mAb 9E10 (10 μg/ml; Roche), followed by two washes andincubation with saturating amounts of FITC-labeled anti-mouse IgG (13μg/ml; Jackson Immuno Research, West Grove, Pa.) for 30 min at 4° C. Toexclude dead cells from the analysis, cells were washed as above andresuspended in FACS buffer containing 10 μg/ml propidium iodide(Sigrna). Background fluorescence was determined by using cellsincubated with 9E10 antibody and FITC-labeled anti-mouse antibody underthe same conditions. Stained cells were analyzed on a FACScan FlowCytometer (BD Bioscience, San Jose, Calif.), and median fluorescenceintensity (MFI) was calculated using the CellQuest™ software (BDBioscience).

For competition experiments, Raji cells were pre-incubated with 200-foldexcess of scFv in FACS buffer for one hour at 4° C. The mAb RFB4(SouthemBiotech, Birmingham, Ala.) was added and cells were incubatedfor an additional hour at 4° C. After two washes with FACS buffer, boundRFB4 was detected using FITC-labeled anti-mouse IgG. Samples wereanalyzed as described above. Inhibition of RFB4 for binding to Rajicells in the presence of competing scFv was determined as percentage ofmaximal MFI of RFB4 in the absence of competing antibodies or presenceof an irrelevant scFv.

Determination of Affinity Constants (Kd)

Affinity measurements were performed as previously described (Benedictet al., J Immunol Methods 201:223-231, 1997) with the followingmodifications: Varying concentrations of antibodies were incubated intriplicate with 5×10⁵ Raji cells at room temperature in FACS buffer fortwo hours. Bound antibodies were detected under the same conditions, asdescribed above. After two final washing steps, cells were fixed in PBScontaining 2% paraformaldehyde for 15 minutes at room temperature andanalyzed by flow cytometry. The MFI was determined as described aboveand background fluorescence was subtracted. Equilibrium constants weredetermined by using the Marquardt-Levenberg algorithm for non-linearregression with the GraphPad Prism version 3.0a for Macintosh (GraphPadSoftware, San Diego, Calif.).

Biophysical Stability

ScFv fragments were incubated at 37° C. in 90% human serum at aconcentration of 12 μg/ml for up to 144 hours. Samples were taken atdifferent time points and stored at −20° C. Binding activity of thesamples to CD22⁺ Raji cells was determined by flow cytometry. The MFIwas determined as described above. Temperature-dependent degradation ofmonomeric scFv variants was determined by incubation of samples at 4° C.or 37° C. in 90% PBS at a concentration of 12 μg/ml for 120 h, followedby analytical gel filtration.

RESULTS

Immunoreactivity and Antigen Affintiy

Flow cytometry analysis revealed a specific binding of all specificitygrafted versions to several CD22⁺ lines and no binding to CD22− celllines (data not shown). Grafting the antigen binding site of mAb RFB4onto the selected frameworks V_(H#)8/V_(L#)19 (variant SG0) was notsufficient to generate a molecule with appropriate antigen bindingproperties. Variant SGI contained two back-mutations—the residuespredicted to be of the highest importance. SGI showed an antibodyconcentration-dependent increase in fluorescence intensity, butsaturation on CD22⁺ tumor cells was not reached at concentrations up to3.3 μM. Two further light chain back-mutations in variant SGII resultedin a moderate binding affinity (Kd 463 nM, FIG. 3, Table II) to CD22.Variant SGIII contained one V_(H) and four V_(L) frameworkback-mutations and had an apparent Kd of 9.8 nM (FIG. 3, Table II). The47-fold increase in affinity of variant SGIII in comparison with SGII iscaused by a single back-mutation of interface framework residueV_(L)-46R→L. Importantly, four out of five back-mutated residues ofvariant SGIII also restored respective human germline sites (Table I).As a consequence, only one of the back-mutations (V_(L)-46R→L) generateda potentially immunogenic site in the humanized antibody SGIII.

In variant SGIV, additional back-mutations were made at V_(H)-79D→Y(which also restored the human germline residue) and V_(L)-71F→Y. Thisvariant had the highest affinity for the target antigen (K_(d) 6.5 nM,FIG. 3, Table II). The back-mutations V_(H)-40V→T and V_(H)-84V→S wereexpected to have only a minor effect on binding. Surprisingly, whenmutated together (variant SGV), this lead to a 1.5-fold decrease inaffinity when compared with SGIV (FIG. 3, Table II). The murine mAb RFB4revealed an apparent of K_(d) of 2 nM (FIG. 3).

Epitope Specificity

The epitope specificity of variants SGIII, SGIV and SGV, which bound tothe target antigen with high affinity, was tested by binding competitionwith mAb RFB4 on living tumor cells by flow cytometry. Incubation ofCD22⁺ Raji cells with a 200-fold molar excess of the specificity-graftedvariants SGIII, SGIV, or SGV, respectively, almost completely preventedmAb RFB4 from binding to the target cells (>95% inhibition; FIG. 4).This indicates that the variants recognize the same CD22 epitope as themurine antibody.

Biophysical Stability

Since scFv antibodies of clinical relevance must be stable at bodytemperature and resistant towards human serum proteases, we assessed thestability of the specificity grafted variants by determination of theiractual binding activity to living tumor cells after incubation in humanserum at 37° C. for various time points. The variants with appropriateantigen binding affinities SGII, SGIII, SGIV, and SGV, respectively,showed exceptional stability with 89%-93% of their initial bindingactivity to tumor cells after a six day incubation period (FIG. 5).Analytical size exclusion chromatography after five days incubation ofsamples from each variant at 37° C. in PBS revealed a decrease ofmonomeric protein fractions between only 4% and 11% (FIG. 6).

Example 5

Generation of an Additional Humanized RFB4 Variant.

An additional scFV variant was constructed based on the identificationof a residue (V_(L)36) within the human V_(L) framework that could beimportant for increased solubility. V_(L)36 belongs to a small set ofconserved sites making interface contacts with corresponding heavy chainresidues. Mutagenesis of V_(L)36 to the murine donor sequence waspredicted to increase the solubility of the humanized scFv's resultingin higher protein expression yield and to possibly further stabilize themolecule. Consequently, the back-mutation V_(L)36Leu→Tyr was introducedinto variant SGIII (which was previously chosen as a good construct formaking scFv-RNase fusion proteins). The stability of this scFv issimilar to SGIII but soluble monomeric protein can be produced withabout 5-fold higher yield.

A dimeric “diabody” molecule (bivalent scFv) made with SGIII wasdifficult to produce in soluble form, thus making the bacterialexpression of fusion proteins generated with this construct verydifficult. In contrast, a diabody made with the SGIII variant wasproduced at 4.5-fold higher level and has a 10-fold higher affinity thanthe mab RFB4.

Example 6

Generation of RNase Fusion Proteins

RNase fusion proteins comprising scFv/diabody-SGIII and the RNasesangiogenin or rapLR1 were constructed. These fusion proteinsspecifically bind to tumor cells and exhibit cytotoxicity.

SUMMARY

These examples show the generation of humanized scFV by grafting thespecificity of the murine anti-CD22 monoclonal antibody RFB4 onto humanframewoks pre-selected for stability from a phage display library.

Grafting the antigen binding loops of the murine monoclonal antibodyRFB4 directly onto the pre-selected human frameworks did not result in ahumanized scFv fragment which retained sufficient antigen binding. Lossof avidity of initial CDR grafted antibodies is commonly observed andthe introduction of additional murine donor residues into the humanacceptor antibody frameworks is often required to maintain thestructural integrity of the antigen binding site and appropriate antigenbinding (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289, 1992;Foote and Winter, J. Mol. Biol. 224:487-499, 1992; Kettleborough et al.,Protein Eng. 4:773-783, 1991; Queen et al., Proc. Natl. Acad. Sci. USA86:10029-10033, 1989; Riechmann et al., Nature 332:323-327, 1988). Inmost cases the potential of human acceptor framework residues tocompromise antigen binding properties is assessed by a computer homologymodel. In the present study, all antigen binding loops adopted knowncanonical structures (except CDR-H3 for which canonical structurescannot yet be defined) of the antigen binding loops (Chothia et al.,supra, 1989). Therefore, a sequence alignment strategy was applied toidentify residues with possible detrimental effects on antigen binding.

After identifying uncommon amino acids within the human acceptorframeworks by alignment to several sequence reference templates thestructural role of each of these residues was examined on the basis ofinformation on antibodies with known crystal structures. The data showthat this procedure allowed a very accurate prediction of the criticalpotential of each identified unusual framework residue to interfere withthe structural integrity of the grafted antigen binding site. As aresult, grafted scFv's SGIII, SGIV, and SGV, respectively, retainedantigen binding comparable to the donor mAb RFB4. The about 3-5-foldlower affinity constants of these scFv variants when compared with themurine parental mAb RFB4 (FIG. 3) most likely reflect avidity loss dueto the monovalency of the constructs. In comparison, a scFv antibodygenerated from the murine monoclonal antibody CC49 with specificity forthe pancarcinoma antigen TAG-72 exhibited an 8-fold lower relativebinding affinity than the corresponding murine IgG and a dimeric F(ab′)2derivative (Milenic et al., Cancer Res. 51:6363-6371, 1991).

Non-covalently associated variable domains of scFv formatted antibodiesfrequently show a high tendency for aggregation. Temperature dependentaggregation and failure to enrich at tumor xenografts of a high affinityscFv fragment with specificity for the epithelial glycoprotein-2 (EGP-2)was shown to be due to its low biophysical stability (Willuda et al.,Cancer Res. 59:5758-5767, 1999). Grafting the antigen binding loops andseveral structurally important framework residues of this murine scFvonto stable human acceptor frameworks, resulted in a humanized scFvfragment with markedly improved biophysical properties. This constructwas able to enrich at the tumor site efficiently with a tumor to bloodratio of 5.25 after 24 h, while retaining the specificity and affinityof the murine scFv. The engineered humanized scFv antibody retained48.3% of its initial binding activity after 20 h incubation in humanserum at 37° C. and revealed no temperature induced degradation at thistime point as demonstrated by analytical gel filtration.

In the present study, a stringent panning procedure of antibody phagedisplay libraries was used to not only enrich for molecules with goodantigen binding properties, but also favor the selection ofbiophysically stable molecules displayed on phage during severalselection rounds. The selection of such stable scaffolds as disclosedherein, which derived from a small patient-specific phage displaylibrary and thus had very limited diversity, was not expected. Theseresults indicated that panning procedures enriched molecules withextraordinary stability and that this stability could be maintained inthe grafts.

In summary, a panel of humanized scFv antibodies was generated bygrafting the specificity of the murine monoclonal anti-CD22 antibodyRFB4 onto frameworks pre-selected for stability from a phage displaylibrary. The constructs exhibit excellent antigen binding and stabilityproperties and can be expected to possess a low immunogenic potential.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

All publications, patents, accession numbers, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

1. A humanized antibody comprising a heavy chain variable region (V_(H))and a light chain variable region (V_(L)), wherein the CDRs of the heavychain and light chain variable regions are from a donor antibody, andwherein the heavy chain variable region framework has at least 80%identity to a framework comprised by an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. 2.The humanized antibody of claim 1, wherein the heavy chain variableregion framework has at leat 90% identity to the framework comprised byan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, and SEQ ID NO:11.
 3. A humanized antibody comprising aV_(H) and a V_(L), wherein the CDRs of the heavy chain and light chainvariable regions are from a donor antibody, and wherein the light chainvariable region framework has at least 80% identity to a frameworkcomprised by an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
 4. The humanized antibody ofclaim 3, wherein the light chain variable region framework has at least90% identity to the framework comprised by an amino sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
 5. Ahumanized antibody comprising a V_(H) framework having at least 80%identity to the framework of SEQ ID NO:1 and a V_(L) framework that hasat least 80% identity to the framework of SEQ ID NO:2.
 6. The humanizedantibody of claim 5, wherein the V_(H) framework has at least 90%identity to the framework of SEQ ID NO:1 and the V_(L) framework has atleast 90% identity to the framework of SEQ ID NO:2
 7. The humanizedantibody of claim 5, wherein the donor CDR sequences are from RFB4 andfurther, wherein the antibody specifically binds to CD22.
 8. Thehumanized antibody of claim 7, wherein the humanized antibody comprisesdonor antibody amino acid residues at positions V_(H)6, V_(L)3, V_(L)40,V_(L)49, and V_(L)46.
 9. The humanized antibody of claim 8, wherein theantibody has a V_(H) and V_(L) amino acid sequence as set forth in SEQID NO:21.
 10. The humanized antibody of claim 9, wherein the antibodyhas an amino acid sequence as set forth in SEQ ID NO:21.
 11. Thehumanized antibody of claim 8, wherein the antibody further comprises adonor antibody amino acid residue at position V_(L)36.
 12. The humanizedantibody of claim 11, wherein the antibody has a V_(H) and V_(L) aminoacid sequence as set forth in SEQ ID NO:22.
 13. The humanized antibodyof claim 12, wherein the antibody has an amino acid sequence as setforth in SEQ ID NO:22.
 14. The humanized antibody of claim 8, whereinthe antibody further comprises a donor antibody amino acid residues atposition V_(H)79 and position V_(L)71.
 15. The humanized antibody ofclaim 14, wherein the antibody further comprises a donor residue atposition V_(H)40 and position V_(H)84.
 16. A humanized antibody of claim1, wherein the antibody comprises an Fc region.
 17. A humanized antibodyof claim 1, wherein the antibody is an scFv.
 18. An isolated nucleicacid encoding a humanized antibody as set forth in claim
 17. 19. Animmunoconjugate comprising an antibody as set forth in claim 1, linkedto a detectable or therapeutic moiety.
 20. An immunoconjugate comprisingan antibody as set forth in claim 8, linked to a detectable ortherapeutic moiety.
 21. The immunoconjugate of claim 20, wherein, theantibody has a V_(H) and a V_(L) sequence as set forth in SEQ ID NO:21.22. An immunoconjugate of claim 20, wherein the moiety is a therapeuticmoiety that is a cytotoxic moiety.
 23. An immunoconjugate of claim 22,wherein the cytotoxic moiety is an enzyme.
 24. An immunoconjugate ofclaim 23, wherein the cytotoxic moiety is an RNase A family member. 25.An immunoconjugate of claim 24, wherein the RNase A is rapLR1.
 26. Animmunoconjugate of claim 22, wherein the cytotoxic moiety is a toxin.27. An immunoconjugate of claim 20, wherein the moiety is a cytokine.28. An immunoconjugate of claim 20, wherein the moiety is a smallmolecule.
 29. An immunoconjugate of claim 20, wherein the moiety is adetectable moiety.
 30. An immunoconjugate comprising an antibody as setforth in claim 11, wherein the antibody is linked to a detectable ortherapeutic moiety.
 31. An isolated nucleic acid encoding animmunoconjugate comprising an antibody as set forth in claim 8 and atherapeutic or detectable moiety, wherein the therapeutic or detectablemoiety is a polypeptide.
 32. A heavy chain variable (V_(H)) chain havinga framework that comprises at least 80% identity to the framework regionof a V_(H) amino acid sequence selected from the group consisting of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, and SEQ ID NO:11.
 33. The V_(H) chain of claim 32,wherein the framework comprises at least 90% identity to the frameworkregion of the V_(H) amino acid sequence.
 34. The V_(H) chain of claim32, wherein the chain has at least 80% identity to the framework regionof the V_(H) amino acid sequence set forth in SEQ ID NO:1.
 35. The V_(H)chain of claim 34, wherein the chain comprises RFB4 CDRs.
 36. A lightchain variable (V_(L)) chain having a framework region that comprises atleast 80% identity to the framework region of a V_(L) amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 andSEQ ID NO:15.
 37. The V_(L) chain of claim 36, wherein the frameworkcomprises at least 90% identity to the framework region of the V_(L)amino acid sequence
 38. The V_(L) chain of claim 36, wherein theframework has at least 80% identity to the framework region of the V_(L)amino acid sequence set forth in SEQ ID NO:2.
 39. The V_(L) chain ofclaim 38, wherein the chain comprises RFB4 CDRs.
 40. An isolated nucleicacid encoding a V_(H) chain of claim
 32. 41. An isolated nucleic acidencoding a V_(L) chain of claim 36.